Antibodies, also denoted immunoglobulins, are normally synthesised by lymphoid cells derived from B-lymphocytes of bone marrow. Lymphocytes derived from the same clone produce immunoglobulin of a single amino acid sequence. Lymphocytes cannot be directly cultured over long periods of time to produce substantial amounts of their specific antibody. However, a process of somatic cell fusion, specifically between a lymphocyte and a myeloma cell, has been shown to yield hybrid cells that grow in culture and produce a specific antibody known as a monoclonal antibody. The resulting hybrid cell is known as a hybridoma. A monoclonal antibody belongs to a group of antibodies whose population is substantially homogeneous, i.e. the individual molecules of the antibody population are identical except for naturally occurring mutations.
The development of monoclonal antibody technology has provided an enormous opportunity for science and medicine in implementing research, diagnosis and therapy. Monoclonal antibodies are e.g. used in radioimmunoassays, enzyme-linked immunosorbent assays, immunocytopathology, and flow cytometry for in vitro diagnosis, and in vivo for diagnosis and immunotherapy of human disease.
Antibodies are grouped into five different types, namely immunoglobulin G (IgG), which is the most prevalent; immunoglobulin A (IgA); immunoglobulin M (IgM); immunoglobulin D (IgD); and immunoglobulin E (IgE). At present, about thirty percent of the biotechnology-derived drugs under development are based on monoclonal antibodies of type G.
The Y-shaped disposition of the structure of the IgG molecule is well known from standard biochemistry textbooks. In brief, regarding its tertiary structure, one intact IgG molecule consists of six globular regions, each of which is formed by two domains. Regarding its primary structure, an IgG consists of two light chains and two heavy chains, which are covalently linked by disulphide bridges. The two globular parts that correspond to the “base of the Y” form the Fc fragment and are formed by domains consisting of only heavy chain residues. Contrary to this, each of the “arms of the Y” constitutes a Fab fragment with two globular parts each. Each of the globular parts in a Fab fragment is formed when one domain from the light chain contacts one domain from the heavy chain. It is well known that the globular part located further away from the centre of the antibody comprises the regions known as the hypervariable regions as well as the antigen-binding site.
By sequence homology, heavy chains of IgGs can be classified into the four types 1, 2, 3 and 4 whereas light chains fall into two types called λ and κ. In humans, about 40% of the IgG molecules carry a light chain of λ type whereas about 60% carry a light chain of κ type. IgGs built up of both light and heavy chains inherit both types of partitionings. Accordingly, one partitioning divides IgGs into four subclasses IgG1, IgG2, IgG3 and IgG4 as compared to the second partitioning which divides IgGs into two subtypes λ and κ. The same type of classification can be applied to antibody fragments like Fab fragments and so called F(ab′)2 fragments, which consist of two Fab fragments connected by a disulphide.
These days, IgGs are generated according to standard techniques in large quantities in cellular expression systems. The most widely used production method includes purification via chromatography, which due to its versatility and sensitivity to the compounds often is the preferred purification method in the context of biomolecules. The term chromatography embraces a family of closely related separation methods, which are all based on the principle that two mutually immiscible phases are brought into contact. More specifically, the target compound is introduced into a mobile phase, which is contacted with a stationary phase. The target compound will then undergo a series of interactions between the stationary and mobile phases as it is being carried through the system by the mobile phase. The interactions exploit differences in the physical or chemical properties of the components in the sample. The interactions can be based on one or more different principles, such as charge, hydrophobicity, affinity etc. In the context of antibodies, affinity chromatography is the most widely utilised purification scheme. More specifically, affinity chromatography is a highly specific mode of chromatography wherein molecular recognition process takes place between a biospecific ligand and a target substance by a principle of lock-key recognition, which is similar to the enzyme binding to a receptor. For a general review of the principles of affinity chromatography, see e.g. Wilchek, M., and Chaiken, I. 2000. An overview of affinity chromatography. Methods Mol. Biol. 147: 1-6.
Lawrence et al (J. F. Lawrence, C. Ménard, M-C Hennion, V. Pichon, F. Le Goffic, N. Durand in Journal of Chromatography A, 732 (1996) 277-281: Use of immunoaffinity chromatography as a simplified cleanup technique for the liquid chromatographic determination of phenylurea herbicides in plant material) describes an evaluation of polyclonal antibodies for cleanup of extracts of food samples. More specifically, antibodies were generated in rabbit after inoculations with an antigen prepared from an urea herbicide. Thus, the antibodies were highly specific to the urea herbicide, which is consequently not useful in any method of general antibody purification.
Another application of urea compounds is provided in EP 0 743 067 (Toray Industries), wherein the compounds are presented as highly selective adsorbing materials used for elimination or detoxification of superantigens from body fluids. The superantigens described are enterotoxins and exotoxins, which are large proteins.
In the field of affinity chromatography, various patents and patent applications relate to protein A, which is an IgG-binding cell wall protein of the bacteria Staphylococcus aureus, and its use as a ligand. For example, PCT/SE83/00297 (Pharmacia Biotech AB) discloses a recombinant form of protein A, wherein a cysteine residue has been added to the protein A molecule to improve its coupling to a separation matrix for subsequent use as an affinity ligand. Further, U.S. Pat. No. 6,197,927 (assigned to Genentech Inc.) discloses Z domain variants of Staphylococcal protein A exhibiting an IgG-binding capacity equivalent to the wild type Z domain, but a significantly reduced size. However, the binding properties of protein A are not ideal. As is well known, protein A binds to IgG molecules from various mammals, with the highest affinity to the human subclasses of IgG1, IgG2 and IgG4. It binds primarily to a surface formed at the juncture of both the second and the third constant domains, known as CH2 and CH3, of IgG located on the Fc fragment. Consequently, protein A cannot be used in affinity purification of any other fragments of IgG than Fc-containing fragments. In addition, even though protein A binds to some Fab fragments, this binding is not generic, since it targets the variable region. However, the interest in Fab and F(ab′)2 fragments has increased lately, since they are smaller than intact IgG molecules but still contain the functional antigen-binding region. Accordingly, the above-mentioned lack of generality becomes another drawback with protein A ligands. Moreover, in attempts to purify IgGs of subclass 3 with protein A-ligands, problems have been reported due to a precipitation of the IgG3 which precipitation is irreversible, thereby causing a loss of purified antibody. Furthermore, protein A exhibits some further drawbacks related to its being a protein. Like most proteins, it is amenable to proteolytic degradation, which may pose serious problems e.g. if a cell lysate is directly applied to a column comprising protein A-based ligand, since most cell lysates will also comprise various proteases. Further, protein A-based ligands are usually labile to the conventionally used cleaning in place (cip) procedures at high pH conditions, which renders reuse of the column more difficult. In addition, protein A-based affinity ligands have also been known to be unstable under acidic conditions, which may result in an undesired leakage of the ligand during the purification process which will both contaminate the product and impair the quality of the purification system.
Another ligand suggested for use in affinity chromatography has been disclosed in U.S. Pat. No. 4,977,247, namely the cell wall protein known as protein G. More specifically, protein G exhibits a different affinity to IgGs as compared to protein A. Protein G binds to a highly conserved region of the constant part of the Fab fragment, primarily to residues from the heavy chain, and consequently it has potential to be used as a generic Fab binder. However, it has been reported that protein G has a reduced binding to Fab fragments of type IgG2. In addition, protein G shares most of the disadvantages of protein-based affinity ligands discussed above in relation to protein A. Furthermore, many of the known protein-based affinity ligands have proven to be relatively expensive to produce.
Consequently, there is a need of novel IgG-binding ligands of a more advantageous nature, which are also more cost-effective to produce. Such new ligands should avoid the above-discussed drawbacks, and preferably also involve more preferable binding properties than the hitherto suggested ligands.
In a recent work by the present inventors, which at the time of filing of the present patent application was still not published, a novel binding site that exhibits the spatial conformation of a pocket was identified. The binding pocket was shown to be specific for human kappa IgGs of all subtypes.
The recently identified binding pocket directed the present inventors to a new target on the human IgG molecule in their efforts to find a new affinity ligand with improved properties as compared to the prior art.